1. Field of the Discovery
The description provides methods of nucleic acid amplification and detection reactions, which are useful for rapid pathogen detection or disease diagnosis.
2. Background Information
Nucleic acid analysis methods based on the complementarity of nucleic acid nucleotide sequences can analyze genetic traits directly. Accordingly, these methods are a very powerful means for identification of genetic diseases, cancer, microorganisms etc. Nevertheless, the detection of a target gene or nucleic acid present in a very small amount in a sample is not easy, and therefore, amplification of the target gene or its detection signal is necessary. As such, in vitro nucleic acid amplification technologies (NAATs) are an invaluable and powerful tool for detection and analysis of small amounts of nucleic acid in many areas of research and diagnosis.
NAAT techniques allow detection and quantification of a nucleic acid in a sample with high sensitivity and specificity. NAAT techniques may be used to determine the presence of a particular template nucleic acid in a sample, as indicated by the presence of an amplification product (i.e., amplicon) following the implementation of a particular NAAT. Conversely, the absence of any amplification product indicates the absence of template nucleic acid in the sample. Such techniques are of great importance in diagnostic applications, for example, for determining whether a pathogen is present in a sample. Thus, NAAT techniques are useful for detection and quantification of specific nucleic acids for diagnosis of infectious and genetic diseases.
NAATs can be grouped according to the temperature requirements of the procedure. For example, the polymerase chain reaction (PCR) is the most popular method as a technique of amplifying nucleic acid in vitro. This method was established firmly as an excellent detection method by virtue of high sensitivity based on the effect of exponential amplification. Further, since the amplification product can be recovered as DNA, this method is applied widely as an important tool supporting genetic engineering techniques such as gene cloning and structural determination. In the PCR method, however, temperature cycling or a special temperature controller is necessary for practice; the exponential progress of the amplification reaction causes a problem in quantification; and samples and reaction solutions are easily contaminated from the outside to permit nucleic acid mixed in error to function as a template (See R. K. Saiki, et al. 1985. Science 230, 1350-1354). Other PCR-based amplification techniques, for example, transcription-based amplification (D. Y. Kwoh, et at. 1989. Proc. Natl. Acad Sci. USA 86, 1173-1177), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA 88, 189-193), and restriction amplification (U.S. Pat. No. 5,102,784) similarly require temperature cycling.
More recently, a number of isothermal nucleic acid amplification techniques (iNAATs) have been developed. That is, these techniques do not rely on thermocycling to drive the amplification reaction. Isothermal amplification techniques typically utilize DNA polymerases with strand-displacement activity, thus eliminating the high temperature melt cycle that is required for PCR. This allows isothermal techniques to be faster and more energy efficient than PCR, and also allows for more simple and thus lower cost instrumentation since rapid temperature cycling is not required. For example, methods such as Strand Displacement Amplification (SDA; G. T. Walker, et at. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396; G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696; U.S. Pat. No. 5,648,211 and EP 0 497 272, all disclosures being incorporated herein by reference); self-sustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci. USA 87, 1874-1878, which is incorporated herein by reference); and Qβ replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202, which is incorporated herein by reference) are isothermal reactions (See also, Nucleic Acid Isothermal Amplification Technologies—A Review. Nucleosides, Nucleotides and Nucleic Acids, 2008. v27(3):224-243, which is incorporated herein by reference).
In the SDA method, a special DNA polymerase is used to synthesize a complementary chain starting from an amplification primer complementary to the 3′-side of a certain nucleotide sequence template, and including one or more bumper primers upstream of the amplification primer to displace the double-stranded chain if any at the 5′-side of the sequence template. Because a double-stranded chain at the 5′-side is displaced by a newly synthesized complementary chain, this technique is called the SDA method. The temperature-changing step essential in the PCR method can be eliminated in the SDA method by previously inserting a-restriction enzyme recognition sequence into an annealed sequence as a primer. That is, a nick generated by a restriction enzyme gives a 3′—OH group acting as the origin of synthesis of complementary chain, and the previously synthesized complementary chain is released as a single-stranded chain by strand displacement synthesis and then utilized again as a template for subsequent synthesis of complementary chain. In this manner, the complicated control of temperature essential in the PCR method is not required in the SDA method.
In the SDA method, however, the restriction enzyme generating a nick should be used in addition to the strand displacement-type DNA polymerase. This requirement for the additional enzyme is a major cause for higher cost. Further, because the restriction enzyme is to be used not for cleavage of both double-stranded chains but for introduction of a nick (that is, cleavage of only one of the chains), a dNTP derivative such as alpha-thio dNTP should be used as a substrate for synthesis to render the other chain resistant to digestion with the enzyme. Accordingly, the amplification product by SDA has a different structure from that of natural nucleic acid, and there is a limit to cleavage with restriction enzymes or application of the amplification product to gene cloning. In this respect too, there is a major cause for higher cost. In addition, when the SDA method is applied to an unknown sequence, there is the possibility that the same nucleotide sequence as the restriction enzyme recognition sequence used for introducing a nick may be present in a region to be synthesized. In this case, it is possible that a complete complementary chain is prevented from being synthesized.
Loop-Mediated Isothermal Amplification (LAMP) is another isothermal nucleic acid amplification technique. In LAMP, the target sequence is amplified at a constant temperature of 60-65° C. using either two or three primer sets, and a polymerase with high strand displacement activity in addition to a replication activity. (See Nagamine K, Hase T, Notomi T (2002). “Accelerated reaction by loop-mediated isothermal amplification using loop primers”. Mol. Cell. Probes 16 (3): 223-9; and U.S. Pat. No. 6,410,278, which is incorporated herein by reference).
LAMP was originally invented and formulated as an isothermal amplification with the strict requirement for four primers: two loop-generating primers (FIP and BIP comprising F1, F2 and B1, B2 priming sites, correspondingly) and two “Displacement primers” (F3 and B3) (FIG. 1). However, in this manifestation the LAMP technology was far too slow for the majority of practical applications. In order to increase the speed of LAMP-based assays the inventors of LAMP came up with additional “Loop primers” which, when added in conjunction with the other primers used in LAMP, resulted in significantly faster assays. Currently, the commonly used manifestation of LAMP requires a total of six primers: two loop-generating primers, two displacement primers and two “Loop primers” (LB and LF).
Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The reaction can be followed in real-time either by measuring the turbidity or by fluorescence using intercalating dyes. Dye molecules intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. Thus, LAMP provides major advantages due to its simplicity, ruggedness, and low cost, and has the potential to be used as a simple screening assay in the field or at the point of care by clinicians.
Primer design for LAMP assays thus requires the selection of eight separate regions of a target nucleic acid sequence (the FIP and BIP primers encompass two primer binding sites each), with the BIP/FIP and Loop primers having significant restrictions on their positioning respective to each other. “Loop primers” must be positioned strictly between the B2 and B1 sites and the F2 and F1 sites, respectively, and must be orientated in one particular direction. Further, significant care must be taken in primer design to avoid primer-dimers between the six primers needed (especially difficult as the FIP and BIP primers are generally greater than 40 nucleotides long). As a consequence, LAMP primer design is extremely challenging, especially when targeting highly polymorphic markers and sequences containing complex secondary structure. Also, because LAMP uses 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of the genome, and because primer design is subject to numerous constraints, it is difficult to design primer sets for LAMP “by eye”. Software is generally used to assist with LAMP primer design, although the primer design constraints mean there is less freedom to choose the target site than with PCR. In a diagnostic application, this must be balanced against the need to choose an appropriate target (e.g., a conserved site in a highly variable viral genome, or a target that is specific for a particular strain of pathogen).
LAMP has been observed to be less sensitive than PCR to inhibitors in complex samples such as blood, likely due to use of a different DNA polymerase (typically Bst DNA polymerase rather than Taq polymerase as in PCR). LAMP is useful primarily as a diagnostic or detection technique, but is not useful for cloning or myriad other molecular biology applications enabled by PCR.
Also, multiplexing approaches for LAMP are relatively undeveloped. The larger number of primers per target in LAMP increases the likelihood of primer-primer interactions for multiplexed target sets. The product of LAMP is a series of concatemers of the target region, giving rise to a characteristic “ladder” or banding pattern on a gel, rather than a single band as with PCR. Although this is not a problem when detecting single targets with LAMP, “traditional” (endpoint) multiplex PCR applications wherein identity of a target is confirmed by size of a band on a gel are not feasible with LAMP. Multiplexing in LAMP has been achieved by choosing a target region with a restriction site, and digesting prior to running on a gel, such that each product gives rise to a distinct size of fragment, although this approach adds complexity to the experimental design and protocol. The use of a strand-displacing DNA polymerase in LAMP also precludes the use of hydrolysis probes, e.g. TaqMan probes, which rely upon the 5′-3′ exonuclease activity of Taq polymerase.
More recently, investigators have developed a modified LAMP technique called, STEM. The LAMP-STEM system utilizes “Stem primers,” which are directed to the stem portion of the LAMP amplicon (or “dumbbell”). Stem primers can be used as an alternative to LAMP “Loop primers” (See FIG. 2). When used in addition to loop-generating and displacement primers, Stem primers offer similar benefits in speed and sensitivity to the Loop primers. (See Gandelman et al., Loop-Mediated Amplification Accelerated by Stem Primers. Int. J. Mol. Sci. 2011, v12:9108-9124, and US 2012/0157326, which are both incorporated herein by reference). This beneficial effect of Stem primers is surprising as they do not bind to the single-stranded DNA loops, which define the very nature of the LAMP technology. Stem primers can be employed in either orientation, do not require either the B2/B1 or F2/F1 sites to be a specific distance apart, can be multiplexed, and allow the F1 and B1 sites to be positioned further from each other than in LAMP.
STEM primers significantly accelerate LAMP comprised of loop-generating and displacement primers only. They can be used on their own or synergistically with other STEM primers or even Loop primers. Addition of Stem primers into LAMP has a positive effect on both speed and sensitivity. In some cases they improve reproducibility at low copy number. The action of Stem primers can be rationalized via the proposed mechanism of LAMP. They anneal to transiently single-stranded regions of the amplicon and recopy the entire binding sites for the BIP/FIP primers. An additional unique feature is the extra strong intra-molecular self-priming when Stem primers delimit amplicon.
In general, positioning of Stem primers is less constrained than that of Loop primers. A rather challenging primer design involving selection of at least eight binding sites is thus simplified. Furthermore, Stem primers impose fewer limitations on the primer design in terms of stem length, orientation and distances between B1-B2 and F1-F2 sites. In contradiction to the postulated LAMP mechanism that relies on the involvement of displacement primers Stem primers can occasionally allow displacement primers not to be used at all, though it is not clear why this is so. This has a major implication for primer design, as it allows the ability to omit one displacement primer or even both, if necessary.
In many circumstances, such as point of care diagnostics, it is advantageous to be able to simultaneously amplify and detect multiple targets in a single sample using a single assay. This is typically done by combining the amplification of multiple targets in the same tube using different dyes attached to each different target primer set or probe. This very common method has two significant drawbacks. One, since all the primers are together in one solution, there is a very high chance of them cross-reacting with each other and creating dimers and other spurious products that would interfere with the results. This is overcome by laboriously screening many combinations of primer sets to find ones that to not cross-react.
The second major limitation of this method is that there are a limited number of dyes that can be separately detected when in the same solution. Since the wavelength of the emitted light from the dye has some bandwidth to it, each dye's emission spectrum must be adequately separated from the others in order for specific and reliable detection. In practice, this limits each amplification reaction to the detection of 5 or 6 different targets.
A technique that could detect a higher number of targets from the same sample without compromising sensitivity would be a huge improvement for many applications, such as a respiratory infection screening panel, where about 20 different targets are required for a thorough test. Another example is Tuberculosis, where about 20 different alleles must be screened in order to accurately and specifically determine the presence of resistance to either a the two front-line drugs.